1. Field of the Invention
The present invention relates to a biological reaction analysis technique that employs an on-chip polarimeter (OCP) to monitor optical activity in samples contained in one or more microfluidic channels formed in a chip or substrate. The sample in each channel is illuminated by a linearly polarized laser beam and the resulting interference fringe pattern in the light scattered by the sample is interrogated with a transducer. The technique is particularly suited for high-throughput analysis.
2. Description of the Background Art
The ability to measure optical activity is essential in the pharmaceutical, food and biotechnology industries. It is particularly important in the development of new drugs, with the vast majority of medicines and drug metabolites being chiral and with the worldwide sale of chiral drugs in single-enantiomer dosages exceeding $147 billion in 2001. Chiral molecules, which exist in pairs of optical isomers called enantiomers that are structurally identical, with the same physical properties, and differ only in their three-dimensional spatial arrangement. In effect, the two enantiomers are just like a pair of hands in that they are non-superimposable mirror images of one another with no planes of symmetry. In the industries, the two isomers are referred to as R and S isomers.
Drugs work by reacting with receptors in the body that have a specific physical shape and usually fit one enantiomer better than the other. In other words, one isomer binds preferentially while the other has little or no activity. While there are examples where both enantiomers have similar therapeutic properties, there are many other cases where one of the isomers causes serious side-effects. As a result, all chiral forms of a drug now have to be tested rigorously for possible side-effects and for chiral stability in vivo before approval. The FDA insists on switching to the pure enantiomer for older drugs and will only approve single isomers of new chiral drugs. It is therefore certain that the percentage of single-enantiomer dosages will grow from the current level of 40% of all drug sales.
With the advent of combinatorial chemical methods and the implementation of parallel synthesis coupled with high-throughput screening, the pace of research in drug discovery has significantly accelerated. A significant number of chiral drug precursors are now produced by enzyme catalyzed processes. Improvements in enzyme specificity and activity are now increasingly being obtained via “directed evolution” methodologies. One of the main challenges of combinatorial asymmetric catalysis and directed evolution is the requirement to screen libraries ranging from 104 to 107 members. Recent reports further demonstrate the importance of producing and analyzing chiral compounds. Several reviews have been provided on the advances in the methodologies for the determination of enantiomeric excesses, noting the significant analytical challenges of all things combinatorial.
While no technique is ideal, progress has been made toward the high-throughput (HT) screening of enantioselective enzymes and biological processes, for the purpose of directed evolution. The most obvious approach, UV/Vis, has been restricted to the hydrolytic kinetic resolution of chiral p-nitrophenol esters catalyzed by lipases or esterases (4,800 samples/day, 9% precision). Fluorescence assays require an active probe attached to the substrate (8,000 samples/day, 10% precision). NMR requires pseudo-enantiomers to be created (1,400 samples/day, 5% precision). Capillary array electrophoresis requires a fluorescence-active reagent (20,000 samples/day, 3% precision) and employs the somewhat tedious CE separation technique. Gas chromatography can provide exact ee determinations but is complicated and limited to volatile compounds with throughput of 700 measurements per day. Circular dichrosim (CD) can also provide exact ee determinations but is limited to 700-900 measurement/day and requires complicated calculations. MS requires a mass-tagged chiral derivatization (be rendered “pseudo-enantiomeric” so the enantiomers differ in mass) agent be applied to the mixture (10,000 samples/day, 2% uncertainty). Among the more fascinating techniques for HT ee determinations, include a color test using chirality-dependent doped films of liquid crystals, fluorescent reporting using a DNA microarray, an assay employing antibodies that is an analog of competitive enzyme immunoassay and a technique termed EMDee which is an enzymatic method for determining enantiomeric excess. These promising techniques are somewhat limited by moderate throughput, the need for sample labeling or large amounts of sample.
Polarimetry, an intrinsic optical technique, can provide a quantitative measure of optical activity or chirality. If a plane of polarized light is passed through a sample of each enantiomer in a pair, one will rotate the polarization of the light to the left (levorotatory or (−)-enantiomer), and the other will rotate the polarization plane to the right (dextrorotatory or (+)-enantiomer). This response is proportional to concentration of the chiral molecule, it can be very sensitive, and is a nondestructive well-established technique. For these reasons a number of groups have worked towards improving the performance of the polarimeter for measuring optical activity while reducing the sample size needed for an assay.
One polarimetry device known as a capillary polarimetric detector (CPD) was developed in 1996 that demonstrated for the first time that polarimetric measurements could detect changes in optical activity at the micromolar level in nanoliter volumes and that these measurements had little sensitivity to refractive index perturbations. The CPD has a simple optical train based on a 4-mW polarized He/Ne laser, a polarizing plate with an extinction ratio of 1:10,000 to further purify the polarization state of the beam, a fused silica capillary tube containing the sample to be analyzed and a transducer for receiving the fringe patterns that are caused by interaction of the laser light with the sample in the capillary. Using the CPD it appears possible to quantify rotation at the level of 9×10−6°. The device has been used to analyze D-β-hydroxybutyrate at the picomole level, for the determination of absolute optical activity and very recently for flowing stream analysis. However, the CPD is not a high-throughput device, nor is it inherently compatible with microfluidics technology. Microfluidics allows higher order system integration and ease in sample multiplexing resulting reduced sample consumption and analysis time. To fully realize the potential of microfluidics, detectors that directly interrogate the sample in the channel on the chip, without loss in sensitivity must be developed.
There appear to be two fundamental limitations in the implementation of all previous approaches based on conventional polarimetry for HT measurements or for use as an on-chip optical activity detector; first they demand very high extinction ratios (possible with Glan-Thompson prisms, but problematic for imaging systems due to a limited NA) so as to measure small optical rotation changes. The most common approach used in HT screening is to illuminate a multiwell plate and use an imaging system to collect the light from all of the samples in the well plate simultaneously. Second, conventional polarimeters have an inherent path length dependency as defined by Biot's law and thus have poor sensitivity for optical activity in the short path lengths of a multiwell plate.